Anthocyanins, a class of polyphenols, are responsible for the blue, red, and purple color in many fruits and vegetables. Increasing evidence shows that anthocyanins are potent antioxidants and are associated with protective effects against many coronary diseases such as cancer, cardiovascular diseases, and even obesity. Interest on the use of anthocyanins, as alternatives to synthetic colors in foods, has increased and many researchers are continuing investigating their potential health benefits. Obtaining high-purity anthocyanins is essential for such research. Many bioassays on anthocyanin-rich commodities would not be feasible without eliminating bioactive impurities that obscure interpretation of results. In the food colorant industry some potential low-cost anthocyanin sources could not be commercialized because of co-extracted adverse flavor or even toxic chemicals. Current anthocyanin separation methods are not practical to achieve high purity at reasonable cost. In this study we attempted to develop a new technique that can substantially elevate anthocyanin purity using a low-cost and high-throughput procedure.
To date there have been over 540 naturally occurring anthocyanins identified. Unfortunately, there are only a limited number of pure standards commercially available at high cost. Therefore, many biological studies are performed using crude anthocyanin extracts from fruits and vegetables. Isolation methods range from simple water or organic solvent extraction to various forms of chromatography. Solid-phase extraction (SPE) methods currently are the most commonly used, due to a balance of efficiency and cost. However, such methods normally rely on hydrophilic or hydrophobic interactions between the sorbent and the analyte, which would inevitably allow for a broad spectrum of plant constituents to mix into the anthocyanin fraction. The impurities, usually phenolic compounds, are likely to have biological effects, as well, and therefore become confounding factors in bioassays. Thus, explanation of anthocyanin bioactivity could be vague, and results from different labs could be hardly comparable given the different isolation methods employed.
Broad Statement
Disclosed is a method for separating anthocyanins depleted in phenolic mixture content from fruits, vegetables, and flowers (herein, collectively, plant tissue) feedstock containing anthocyanins and phenolic mixtures. The first step is to contact the feedstock with a mixed-mode cation-exchange resin at low pH for a time period effective for the resin to selectively bind with the anthocyanins and other phenolics. Next, the non-anthocyanin phenolic mixture is selectively separated from the resin by solvent wash for recovery. The resin is subjected to additional solvent wash to release the anthocyanins for recovery. For human consumption, the solvent should be a food-grade solvent, i.e., a solvent permitted by regulation for human consumption. For animal (excluding humans) consumption, the solvent should be an animal-grade solvent, a solvent permitted by regulation for animal (non-human) consumption.
Advantages of the process disclosed herein include the successful use of mixed-mode cation exchange for anthocyanin purification, which is believed to function due to the use of a combination of cation exchange and hydrophobic interaction. Another advantage is the achievement of higher purity than current methodology for fractionation of anthocyanins at comparable cost. A further advantage is the ability to purify the same amount of anthocyanins using much less organic solvents than prior purification processes with less processing time being required. The lifetime and consistency of this polymer-based resin also exceed the conventional silica based resin and therefore result in reduced cost and improved reproducibility.